Like all retroviruses, human immunodeficiency virus (HIV) requires reverse transcription of the viral RNA genome as the first step of replication. Currently approved anti-HIV drugs are aimed at inhibiting either reverse transcriptase (RT) or the viral protease both of which are required for reverse transcription. In response to these drugs, the virus has a propensity to mutate, resulting in resistance. Thus, the development of new strategies and targets for inhibition of HIV-1 replication is essential. One aspect of reverse transcription that has not been investigated for the-development of antivirals is the initiation step. The substrate of RT, the initiation complex, is formed by the association of the viral genomic RNA with a host primer tRNA (tRNALys,3). The initiation complex is a potential nonprotein target for anti-HIV drugs. Development of drugs to prevent formation of this complex or to prevent its recognition by RT might result in enhanced inhibition of reverse transcription, reducing the potential for the generation of drug resistant mutants. We propose to determine the structure of the HIV-1 initiation complex by a combination of in vitro and in vivo studies, using molecular modeling, chemical and enzymatic probing, high resolution NMR, and genetic studies using infections HIV- 1 proviruses. The data from the experiments will be incorporated into three-dimensional models. In turn, the models will provide structural hypotheses that will help establish the priorities of subsequent experimental work. A unique feature of this Program Project is the availability of genetic and molecular biological methods that can test the biological relevance of the in metro structural studies. This approach for determining the structure of the HIV-1 initiation complex offers a solid foundation for the development and testing of a new class of drugs that would inhibit the initiation of reverse transcrip- tion, a critical step in HIV-1 replication.